A fluorescence detecting device that receives fluorescence from the fluorochrome of the object to be measured by irradiating the object to be measured with the laser beam to identify the type of the object to be measured is employed in the flow cytometer used in the medical or biological field.
Specifically, the flow cytometer is a device that mixes samples such as cells, DNAs, RNAs, enzymes, proteins, or micro beads, which are labeled as the fluorochrome, with a normal saline to produce a sample solution by using the binding of antigen-antibody reaction and the like. Then, the device allows the sample solution to flow so as to be encompassed with another solution called “sheath solution”, resulting that a laminar sheath flow in which the labeled samples flow at a speed of about 10 m or lower per second is formed while a pressure is exerted on the laminar sheath flow. The device irradiates the sheath flow with a laser beam to measure the fluorescence or a scattered light in each of the samples. For example, in the case where a variety of samples are to be analyzed, the flow cytometer measures the fluorescent intensity of the fluorescence that is generated by the sample, and identifies which sample having which fluorescence characteristics has passed by among the many kinds of samples. In this case, labeled samples to which fluorochromes of different kinds are adhered are used for the many kinds of samples. The flow cytometer irradiates the labeled sample with the laser beam, and measures the fluorescence that is generated with the irradiation of the laser beam.
Further, the flow cytometer is capable of measuring an intracellular relative quantity of, for example, DNAs, RNAs, enzymes, or proteins within a cell, and also analyzing activity thereof in a short time. Further, there is used a cell sorter that specifies a specific type of cell or chromosome by fluorescence, and selectively collects only the specific cell or chromosome in a live state in a short time.
In the use of the cell sorter, it is required to specify more objects to be measured from information on fluorescence in a short time.
In the following Non-patent Document 1, there is disclosed a flow cytometer that irradiates fluorochromes with plural laser beams that are different in the wavelength band such as 488 nm, 595 nm, and 633 nm, separates plural kinds of fluorescence that are different in the wavelength band which are generated from the fluorochrome by the respective laser beams by using a band pass filter, and detects the separated fluorescence by means of a photoelectron multiple tube (PMT). With the above configuration, it is possible to identify the fluorescence from the plural fluorescent reagents (fluorochrome) to specify the plural kinds of objects to be measured at once.
However, although the wavelength range of the fluorescence that is generated from the fluorescent reagent has a relatively wide width such as about 400 to 800 nm, only 3 to 4 wavelength bands of the fluorescent reagent can be effectively used for an identifiable labels in the visible wavelength range. An increase in the number of fluorescence that can be identified by employing the plural fluorescent reagents is restricted.
Further, in order to increase the number of identifiable fluorescence, it is possible to increase the number of identifiable fluorescence by using the wavelength of fluorescence as well as the intensity of detected fluorescence. However, even in this case, the number of identifiable fluorescence by using the intensity of fluorescence is about 2 to 5, and even if this number is combined with the above-mentioned number of identifiable fluorescence of about 3 to 4 wavelength bands, the number of identifiable fluorescence is about 20 at maximum. For that reason, there arises such a problem that it is difficult to identify and analyze an extremely large number of objects to be measured in a short time even if the above flow cytometer is used.
For example, in the case where a biologic material such as DNA is analyzed by the flow cytometer, fluorochrome is adhered to the biologic material with a fluorescent reagent in advance, and the biologic material is labeled by a fluorochrome different from fluorochromes that have been adhered to micro beads which will be described later. Then, the biologic material is mixed with a solution containing micro beads of 5 to 20 μm in diameter, the micro beads having a surface provided with a unique structure such as carboxyl group. The structure of carboxyl group acts on a biologic material of a certain known structure, and conducts a biological coupling therewith. Accordingly, in the case where the flow cytometer detects fluorescence from the micro beads and fluorescence of the biologic material at the same time, it is found that the biologic material is biologically coupled with the structure of the micro beads. As a result, it is possible to analyze the characteristic of the biologic material. However, in order to analyze the characteristics of the biologic material in a short time by providing various micro beads having various coupling structures, an extremely large number of kinds of fluorochromes are required. However, since the number of kinds of fluorescent reagents identifiable at the same time is small, it is impossible to analyze the biologic material efficiently in a short time by using a large variety of micro beads at once.
Further, there is proposed a method in which plural measurement points at which an object to be measure is irradiated with laser beams to measure fluorescence are provided in the longitudinal direction of a flow tube, and the laser beams irradiated at the respective measurement points are prevented from interfering with each other. However, in this case, it is necessary to provide a large number of laser beams and a large number of light receiving sections in correspondence with the number of measurement points. Further, since a flow tube that forms the flow cell is elongated, the flow path resistance of the sheath solution that flows in the tube becomes large, and a pressure to be exerted on the sheath solution becomes large. For that reason, there arises such a problem that the device is increased in size.
Further, when fluorescence is measured by using the flow cytometer, it is necessary to measure and identify the fluorescence generated by a large variety of fluorochromes and the autofluorescence generated by the samples per se such as the cells or the micro beads at the same time. For that reason, the flow cytometer is equipped with plural photoelectric converters that are different in received light wavelength band from each other, and the fluorochromes that match the received light wavelength bands are selected and used, respectively. In this situation, the measured values that have been obtained by the plural photoelectric converters represent the fluorescence intensities in the respective fluorochromes. However, when the plural kinds of fluorescence are received in the received light wavelength range of the photoelectric converters at the same time, the measured results of the fluorescence intensities are deviated from the actual fluorescence intensities. In order to correct this deviation, the detected values are generally corrected.
As the above correction, for example, there is disclosed a fluorescence value correcting method in the following Patent Document 1.
According to the following Patent Document 1, the measured values that have been obtained by the plural photoelectric converters are represented as vectors. On the other hand, the inverse matrix of a predetermined correction matrix is produced, and the produced inverse matrix acts on the above vectors, thereby enabling the real fluorescence intensities to be calculated. In this case, as shown in FIGS. 8B and 9B of Patent Document 1, the correction matrix is a matrix of geometric transformation which corrects the positional relationship in a two-dimensional correlation diagram (scattergram). For that reason, in order to produce the inverse matrix from the correction matrix and make the inverse matrix act on the vectors having the measured values as vector elements, it is required that the correction matrix is a square matrix. The matrix size of the correction matrix is determined according to the number of photoelectric converters that output the measured values, and a sum of the number of kinds of the autofluorescence generated by the samples (objects to be measured) such as the micro beads or the cells and the number of kinds of the fluorescence generated by the fluorochromes adhered to the samples. As a result, in order for the correction matrix to be the square matrix, the number of photoelectric converters must be made equal to the number of received fluorescence. In other words, when there are provided four kinds of fluorochromes that are adhered to the samples such as the micro beads or the cells, the measurement needs to be conducted by a total of those four fluorochromes and one kind of autofluorescence, that is, five photoelectric converters. The measurement using a large number of photoelectric converters causes an increase in the number of arranged photoelectric converters as well as an increase in the number of processor circuits that process the measured values. As a result, the costs of the flow cytometer and the processing devices are increased. For that reason, there arises such a problem that the kinds of identifiable fluorescence that are measured at the same time are restricted in the number according to the number of arranged photoelectric converters.
Non-patent Document 1: http://www.bdbiosciences.com/pharmingen/protocols/Fluorochrome_Absorption.shtml (searched on Jan. 23, 2005)
Patent Document 1: JP-A-2003-83894